Lipid modified triblock PAMAM-based nanocarriers for siRNA drug co-delivery

Abstract

RNA interference by small interfering RNA (siRNA) holds promise to attenuate production of specific target proteins but is challenging in practice owing to the barriers for its efficient intracellular delivery. We have synthesized a triblock co-polymeric system, poly(amidoamine) dendrimer (generation 4)-poly(ethylene glycol)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (G(4)-D-PEG-(2K)-DOPE). G(4)-PAMAM dendrimer was utilized as a cationic source for efficient siRNA condensation; DOPE provided optimum hydrophobicity and compatible cellular interaction for enhanced cell penetration; PEG rendered flexibility to the G(4)-D for easy accessibility of siRNA for condensation; PEG-DOPE system provided stable micellization in a mixed micellar system. G(4)-D-PEG-(2K)-DOPE was incorporated into the self-assembled PEG-(5K)-PE micelles at a 1:1 molar ratio. Our results demonstrate that the modified dendrimer, G(4)-D-PEG-(2K)-DOPE and the micellar nanocarrier form stable polyplexes with siRNA, shows excellent serum stability and a significantly higher cellular uptake of siRNA that results in target protein down-regulation when compared to the G(4)-PAMAM dendrimer. Moreover, the mixed micellar system showed efficient micellization and higher drug (doxorubicin) loading efficiency. The G(4)-D-PEG-(2K)-DOPE has the higher efficacy for siRNA delivery, whereas G(4)-D-PEG-(2K)-DOPE/PEG-(5K)-PE micelles appear to be a promising carrier for drug/siRNA co-delivery, especially useful for the treatment of multi-drug resistant cancers.

Figure 1

(A) TEM images of G(4)-PAMAM-D and G(4)-D-PAMAM-PEG-DOPE. (B). ¹H-NMR spectra of PEG-DOPEG(4)-D-PEG-DOPE and two starting materials, G(4)-PAMAM-D and NPC-PEG-DOPE recorded in methanol.

Figure 2

Binding ability of tested nanoparticles to siRNA. Different ratios of nitrogen in G(4)-D to phosphate in siRNA (N/P) of the nanoparticles were tested by gel retardation assay (A) and ethidium bromide exclusion assay ((B) and (C)). (D) Evaluation of cytotoxicity of the nanoparticles in C166 and A549 cells after 24 h of incubation.

Figure 3

Evaluation of loading efficiencies of polymers and mixed micellar system. (A) Determination of critical micelles concentrations by pyrene incorporation method. (B) Determination of doxorubicin-loading efficiency of the nanoparticles.

Figure 4

Cellular uptake of nanoparticles (FITC-labeled) in A549 cells. Cells were incubated with 1.5 µM of polymers for 2 h for visualization by confocal microscopy (A) and for 1h and 4h time period for analysis by flow cytometry (B and C). Trypan blue was added for the purpose of quenching the surface associated fluorescence (C). (D) Selected images of the cells in the XY plain at consecutive Z-axis (Z-2,4,6,8).

Figure 4

Cellular uptake of nanoparticles (FITC-labeled) in A549 cells. Cells were incubated with 1.5 µM of polymers for 2 h for visualization by confocal microscopy (A) and for 1h and 4h time period for analysis by flow cytometry (B and C). Trypan blue was added for the purpose of quenching the surface associated fluorescence (C). (D) Selected images of the cells in the XY plain at consecutive Z-axis (Z-2,4,6,8).

Figure 5

Evaluation of siRNA delivery efficiency of the polymers and the mixed micellar system. (A) Representative histogram plot, obtained from fluorescence-activated cell sorting analysis, showing the uptake of Cy5 labeled siRNA, condensed with polymers and micellar system at N/P= 10 after 2 h of incubation with A549 cells. (B) Cellular uptake/delivery of Cy5-labeled siRNA-dendriplxes, measured by the geometric mean of fluorescence, obtained from FACS analysis at 1 h and 4 h time points. (C) Representative dot plot, obtained from FACS analysis, showing the cells labeled with Cy5-siRNA, delivered by various siRNA-dendriplexes. (D) The confocal laser scanning microscope (CLSM) images of siRNA-dendriplexes-dosed A549 cells, after incubation for 2 h. Cell nuclei were stained with Hoechst 33342. Both FACS and microscopy studies were performed at siRNA concentration 100 nM (1:1 mixture of Cy5-labeled siRNA and scramble siRNA) at N/P ratio of 10.

Figure 5

Evaluation of siRNA delivery efficiency of the polymers and the mixed micellar system. (A) Representative histogram plot, obtained from fluorescence-activated cell sorting analysis, showing the uptake of Cy5 labeled siRNA, condensed with polymers and micellar system at N/P= 10 after 2 h of incubation with A549 cells. (B) Cellular uptake/delivery of Cy5-labeled siRNA-dendriplxes, measured by the geometric mean of fluorescence, obtained from FACS analysis at 1 h and 4 h time points. (C) Representative dot plot, obtained from FACS analysis, showing the cells labeled with Cy5-siRNA, delivered by various siRNA-dendriplexes. (D) The confocal laser scanning microscope (CLSM) images of siRNA-dendriplexes-dosed A549 cells, after incubation for 2 h. Cell nuclei were stained with Hoechst 33342. Both FACS and microscopy studies were performed at siRNA concentration 100 nM (1:1 mixture of Cy5-labeled siRNA and scramble siRNA) at N/P ratio of 10.

Figure 6

Efficiency of down-regulating the target protein by siRNA-polymer dendriplexes. Green fluorescence protein-silencing siRNA (siGFP) (200 nM) was delivered to the C166-GFP cells (stably expressing GFP) via dendriplexes in polymer systems (at N/P ratio=10). (A) Geometric mean of fluorescence of the siGFP-treated cells (obtained from the histogram statistics in FACS analysis) was plotted compared to the siNegative treated cells. (B) Fluorescence micrograph, demonstrating the GFP-protein downregulation effect of siGFP treatment. The nuclei was stained with Hoechst 33342.

Figure 7

Dox-delivery efficiency of the nanoparticles analyzed by flow cytometry. (A) Representative histogram plot, demonstrating the differences in the dox-labeling of the A549 cells treated with a fixed dose of dox (4 µg/mL) for 1 h, loaded in the nanoparticles. (B) Quantitative comparison of dox-loaded nanocarrier mediated dox-delivery. Mean fluorescence of the treated cells (obtained by analyzing the histogram statistics) was plotted compared to control.

Figure 8

Assessment of co-delivery efficiency of nanoparticles by flow cytometry. (A) Quantitative comparison of geometric mean of fluorescence from Dox and FAM-labeled siRNA, delivered to the A549 cells via Dox-loaded dendriplex systems. (B) Representative dot plot, obtained by FACS analysis, showing the difference in the Dox- and FAM-siRNA-labeling in the cell populations.

Scheme 1

(A) Schematic representation of the formation of mixed micellar system with G(4)-PAMAM-D-PEG-DOPE/PEG-DOPE. (B) Synthetic scheme of G(4)-PAMAM-D-PEG-DOPE.